This renewal application to study the structure and function of the major histocompatibility complex (MHC) class II genes in vivo focuses on a recently produced strain of class II MHC-deficient mice. We have recently been successful in producing mice deficient for all class II MHC expression, using the approach of gene disruption in embryonal stem cells. It is our goal to use the class II-deficient mice in ways that will allow us to study the structure and function of class II molecules in T cell development, antigen presentation and processes of positive and negative selection, to evaluate the role of the cytoplasmic domain in vivo, and to determine the function of different cellular populations as antigen- presenting cells in vivo. This involves using these mice as recipients for A beta transgenes which have been altered by mutagenesis, or are driven by heterologous promoters which target to selected tissues. We also wish to use them as mating partners to produce and study MHC class I and II double- deficient animals. We have three Specific Aims. In Aim 1, we wish to explore the role of the A beta cytoplasmic domain in T cell-mediated responses, antigen presentation and transmembrane signalling, as a continuation of studies currently funded by this grant. Introduction of an A beta transgene with a truncated cytoplasmic domain into the class II- deficient mice whose only class II molecule will be an I-A molecule formed by the pairing of the endogenous wild-type Aalpha b with the mutated truncated Abeta transgene. The ability of these transgenic mice to respond to foreign protein antigens, tumor antigens, viral antigens and allogeneic and xenogeneic antigens can then be evaluated. The role of the CD 28/B7 pathway and the integrity of transmembrane signalling will also be examined. In Aim 2, we will determine which cell types are involved in antigen presentation, positive selection and tolerance by selective targeting of class II. By using the class II-deficient mice as recipients for an Abetab transgene driven by selected promoters, or as mating partners to existing I-E transgenics, tissue-specific expression of class II in B cells, macrophages and epithelial cells should by possible. In Aim 3, will produce and analyze MHC class I and II double deficient mice by mating the class II-deficient mice with the homozygous Beta2 microglobulin-deficient 129/Sv strain developed by Jaenisch and colleagues. The analysis of these double-deficient mice will be similar to the analysis of the mice expressing a tailless I-A molecule, with special emphasis on the use of these double-deficient animals for solid organ and islet cell transplantation.